Overview of research progress and application of experimental models of colorectal cancer

Colorectal cancer (CRC) is the third most common malignancy in terms of global tumor incidence, and the rates of morbidity and mortality due to CRC are rising. Experimental models of CRC play a vital role in CRC research. Clinical studies aimed at investigating the evolution and mechanism underlying the formation of CRC are based on cellular and animal models with broad applications. The present review classifies the different experimental models used in CRC research, and describes the characteristics and limitations of these models by comparing the research models with the clinical symptoms. The review also discusses the future prospects of developing new experimental models of CRC.


Cellular models based on intestinal cells and CRC cells
In vitro models of CRC established using intestinal cells and CRC cells are frequently employed for obtaining rapidly growing cellular models of CRC and for facilitating experimental control. In vitro models of CRC can simultaneously generate several populations of homogeneous cells. Specific cellular targets of macroscopic systems can be conveniently studied using these models by analyzing the experimental results (Saeidnia et al., 2015).
The first mammalian cell line was established in 1943, which served as a prelude to in vitro cell culture. The CoLo 205 CRC cell line was established in 1957, which promoted in vitro studies on CRC. Figure 1 depicts the history of development of in vitro models of CRC (Sanford et al., 1948;Ricci et al., 2007;Sharma et al., 2010;Jedrzejczak, 2017).

Two-dimensional (2D) cellular models of CRC
CRC cell lines are in vitro tumor models with different origins and types, and serve as fundamental tools for investigating the biomarkers of drug sensitivity, resistance, and toxicity. CRC cell lines are established by isolating CRC cells from patients or animals with CRC followed by culture on artificial media. The appropriate cell lines are selected based on the type of cancer or gene expression levels, according to the aims of the study. SW620, Caco-2, RKO, SW480, HT8, HT29, HT116, LoVo, and LS174 T cell lines are currently widely used in basic research studies on CRC (Akashi et al., 2000;Vécsey et al., 2002;Lind et al., 2004;Barretina et al., 2012;Ahmed et al., 2013;Gemei et al., 2013;Mouradov et al., 2014;Maletzki et al., 2015;Boot et al., 2016;Berg et al., 2017;Mooi et al., 2018;Kim et al., 2020;Bian et al., 2021).
Although the characteristics of CRC cell lines are highly consistent with those of human cancer models, they have certain limitations. CRC cell lines facilitate the investigation of the molecular and phenotypic characteristics of CRC. However, as only one side of the cells is in contact with the medium during culture, the majority of cells gradually flatten, undergo abnormal division, and lose their differentiation phenotype following isolation from tissues and plate culture. Additionally, CRC cells continue to proliferate in vitro, which may cause the cell lines to lose the characteristics of the original tumor. Another limitation of CRC cell lines is the scarcity of matrix ingredients in the tumor microenvironment (TME), including the cells and acellular components constituting the structural complexity of the in vivo environment. Altogether, these indicate that CRC cell lines fail to accurately mimic the in vivo growth characteristics of tumor cells.

Three-dimensional (3D) cellular models of CRC
Owing to the limitations of 2D cellular models of CRC, researchers are committed towards exploiting novel and physiologically representative models of CRC. In vitro 3D culture models, including spheroids and organoids, are therefore used for overcoming the limitations of 2D cellular models. Spheroids comprise a mixture of single-cell or multicellular systems, while organoids are generally formed of specific stem cells or ancestral cells from organs (Kimlin et al., 2013;Boucherit et al., 2020). Spheroids and organoids are superior at mimicking tumor cell heterogeneity and the complex interactions among different cells (Thoma et al., 2014).    , 2020). MCTS models are similar to solid tumors in terms of the growth kinetics, metabolic rate, and resistance to chemotherapy and radiotherapy in vivo (Ivascu and Kubbies, 2006), and have been employed for screening and evaluating the efficacy of drugs. However, the variability of MCTS models makes it difficult to obtain repeatable and stable experimental data, which affects the use of these models in tumor research.
The tumorosphere model of CRC stem cells (CSCs) was used in the early 2000s for evaluating the differentiation capacity of tumors. However, because there are no morphological phenotypes associated with the phenotypic instability of CSCs, the tumorosphere model is unable to faithfully simulate the in vivo 3D framework and physiological condition of tumors (Valent et al., 2012).
The TDTS models consist of cancer and stromal cells, and are commonly used in studies on CRC. TDTS models of CRC tumors have a unique histological feature similar to the poorly differentiated globules produced by permanent cancer cell lines, and can fully simulate the characteristics of in vitro 3D cell culture models of CRC (Santini and Rainaldi, 1999;Weiswald et al., 2009).
OMS models are enriched in stem cells which can represent the complexity of parental tumor cells similar to in vivo tissues by forming an extracellular layer of epithelioid cells and an intracellular layer of mesenchymal cells, and thus maintaining the multicellular nature of CRC (Rajcevic et al., 2014). However, the difficulty of producing homogeneous spheres in a reproducible manner combined with the insufficiency of stable experimental data can prove to be a challenge during the application of the OMS model in CRC research and drug development.

FIGURE 4
Application of CRC cellular models.

FIGURE 5
Chemically induced CRC animal models.

Induced colon and small intestinal carcinogenesis
Requires multiple administration, low specificity Reddy and Mori (1981) Reddy (1998) CIN  Rygaard and poulsen (1969) Lehmann et al. (2017) Frontiers in Pharmacology frontiersin.org human CRC tumors in human intestinal stem cell medium (HISC), and the phenotype and genotype of the PDOs have been reported to be highly similar to those of the original tumor (Van et al., 2015;Vlachogiannis et al., 2018). Organoids are typically used for investigating the mechanism underlying the development of CRC, screening anti-CRC drugs, and determining the efficacy and mechanism of action of drugs. However, there are various limitations to the application of organoids in studies on CRC, which are described hereafter. First, the current methods for organoid culture lack the technological means for maintaining the blood vessels, immune system, and peripheral nervous system of tumor cells, and organoids lacking these characteristics cannot be used in CRC research (Bredenoord et al., 2017). Second, as PDO models lack the cellular and acellular components of the TME of the original tumor, they cannot equivalently represent the in vivo environment of the tumor . Third, there are no specific media for culturing organoids to date. Furthermore, it is unclear whether organoids can represent the overall heterogeneity of the tumor and all cell types in the tumor. Organoids can be applied to relevant studies by optimizing the culture conditions for maintaining the expression of genes related to microsatellite instability, B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations, poor differentiation, or mucinous phenotypes related to CRC. The application of organoids to CRC research can be improved by employing the co-culture model of organoids in which immune cells and mesenchymal cells are co-cultured for simulating the in vivo TME.

Application of cellular models of CRC
The establishment of models using the corresponding tumor cells is crucial for investigating the mechanism underlying the development of CRC and discovery of anti-CRC drugs (Senga and Grose, 2021). The applications of different cellular models of CRC according to the different molecular mechanisms underlying tumor formation, including epithelial-mesenchymal transition (EMT), apoptosis, invasion, metastasis, chromosome instability (CIN), and immune escape, are summarized in Table 1 and Figure 4.  (2021) Groll et al. (2021) Sus scrofa (Pig) The anatomical structure of the small intestine is similar to that of humans. Model has a moderate size and long life. The progression and accumulation of mutations in CRC can be monitored by colonoscopy screening The model cannot be used to study acute CRC as the process of cancer formation is slow

CRC animal models based on experimental animals
The occurrence of diseases such as cancer that occur spontaneously in animals is largely attributed to genetic diversity and immune functions. Therefore, studying the methods for generating animal models of CRC can aid in elucidating the mechanisms underlying the development of cancer (Marian, 2004). Animal models can compensate for the limitations of cellular models that are incapable of simulating the mechanism underlying the development of CRC. Rat and murine models are the most frequently used animal models of CRC, and other animal   LPS tail vein and peritoneal injection + hypodermic implantation of C26 tumor cells to establish colorectal tumor-bearing with syndrome of heat-toxicity and blood stasis model Li et al. (2017b) Frontiers in Pharmacology frontiersin.org models of CRC, including fruit fly, zebrafish, and pigs, are also commonly used as sentinels and preclinical models in CRC research.

Rodent models
Rodent models are conducive tools for conducting cancer research, and are extensively used for elucidating the etiopathogenesis and molecular mechanisms underlying the development of CRC. Previous studies have demonstrated that the protein-coding genes of mice and humans share high homogeneity (Mouse Genome Sequencing Consortium, 2002). Additionally, the use of murine models is advantageous owing to the fact that mice have a short intergenerational interval, high reproducibility, and similar genetic background and formula as humans, compared to other animal models. Murine models of CRC can therefore be used as effective tools for studying the mechanism underlying the pathogenesis of CRC and determining novel strategies for the prevention and treatment of CRC (Doyle et al., 2012).
Transgenic mice models can serve as effective tools for preclinical evaluation and screening during the optimization and development of anticancer drugs. Mutations in APC (adenomatous polyposis coli) are commonly inherited in adenoma-carcinoma transitions observed during the development of CRC (Van et al., 2000). Additionally, the absence of mutations in DNA mismatch repair (MMR) genes increases deletion mutations in APC, which accelerates the formation of adenomas (Huang et al., 2004). It has been reported that mutations in tumor protein 53 (p53), Kirsten rats arcomaviral oncogene homolog (KRAS), phosphatidylinositol-4,5bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), F-box and WD repeat domain containing 7 (FBXW7), SMAD family member 4 (SMAD4), transcription factor 7-like 2 (TCF7L2), NRAS protooncogene (NRAS), AT-rich interaction domain 1 A (ARID1A), SRY-box transcription factor 9 (SOX9), and APC membrane recruitment protein 1 (FAM123B) can also increase the risk of CRC (Cancer Genome Atlas Network, 2012). Transgenic murine models are extensively used for studying the occurrence and elimination of tumors, underlying molecular pathways, and genomic regulation via gain-of-function or loss-of-function mutations in oncogenes and cancer suppressor genes.
CRC is caused by various risk factors, including poor dietary habits, environment, exposure to carcinogenic chemicals, and other factors (Hecht, 2003;Mehta et al., 2017). Animal models of CRC generated by treatment with chemicals serve as effective models in studies aimed at determining novel therapeutic approaches and investigating the diagnosis, prognosis, and identification of predictive markers. The differences among the methods and duration of treatment for inducing CRC with different chemical agents are depicted in Figure 5.
The use of chemical agents for generating models of CRC requires a long duration and these models have longer experimental cycles. Mofikawa et al. established the first orthotopic transplantation model of CRC in 1986 by transplanting human CRC cells under the cecal wall of nude mice. This shortened the period of study using animal models of CRC, and initiated the establishment of tumor transplantation models. Table 2 summarizes the different murine models of CRC, and describes their scope of application and limitations in tumor research.

Other animal models of CRC
In addition to rodents, invertebrates such as fruit fly can be used for personalized diagnosis and developing potential therapeutic strategies for CRC. Vertebrates such as zebrafish, dogs, cats, pigs, and non-human primates are also used in studies on CRC. The advantages and disadvantages of the different animal models used in CRC research are summarized in Table 3.

Application of animal models of CRC
The carcinogenesis of CRC is affected by several contributing factors. The selection of the animal model of CRC depends on the purpose of the study, as summarized in Table 4.
Traditional Chinese medicine (TCM) and western medicine are two different medical theoretical systems. The research model based on the etiological mechanism theory of TCM is applied to animal studies with TCM syndrome, as shown in Table 5.

Conclusions and future directions
Understanding the inherent advantages and limitations of the different models of CRC, and the appropriate application of these models in drug development and studies on the mechanism of tumor occurrence and development are important in CRC research.
Human cell lines and xenograft models have been extensively employed over the past few decades owing to their low cost and ease of application. However, these models are incapable of reproducing the heterogeneity of CRC tumors (Harma et al., 2010). The cell coculture technique can overcome the limitations of monolayer cell culture, and enables the construction of in vitro physiological or pathological models that closely represent the in vivo condition, and can be used for studying the interactions between cells, and between cells and the culture environment. It has been reported that 3D models can mimic the physiological characteristics of parental tumors, including tumor heterogeneity (Li et al., 2019). However, the shape, size, and activity of organoids are different under the same culture conditions, and the matrix limits the penetration of drugs and hinders drug screening (Zhao et al., 2020). It is therefore imperative to construct a model that closely represents the characteristics of CRC in vivo.
The intestinal microarray platforms used in CRC research, which consist of intestinal organoids and organic chips, can summarize the important structural features and functions of the natural duodenum. This platform can be applied for studying drug conveyance, metabolism, and drug-drug interactions (Kasendra et al., 2018). Multi-locus transfer chips consist of multiple 3D organoids that connect the CRC-like organs, liver, lungs, and endothelial flow via recirculating fluid systems, and enables cell tracking by fluorescence imaging technology. The transfer sites of CRC cells are also included in multi-locus transfer chips (Aleman and Skardal, 2019).

Frontiers in Pharmacology frontiersin.org
Animal models of CRC have been widely used for studying the complexity of CRC. There are primarily two types of animal models, namely, in situ models and the cell and tissue transplantation models of CRC. Owing to the relatively simple modeling approach of human tumor xenotransplantation, this model is presently widely used for studying the efficacy of anti-CRC drugs. The effects of CRC xenotransplantation can be closely related to clinical activity via the rational application of these models. For instance, genetically engineered murine models have been used for studying the progression of tissue-specific molecular changes in CRC by determining the effect of specific molecular targets. Chemical induced-CRC animal model is one of the most commonly CRC models, in which CAC model is usually induced by AOM/DSS to study the mechanism of inflammation related-tumorigenesis and development (Zeng et al., 2022). The CRC model with TCM syndrome is an artificial disease and syndrome experimental animal model created by simulating and replicating characteristics of human disease prototype according to TCM theory. An animal model combining with CRC and TCM syndromes might be useful to mimic the clinical characteristics of CRC patients with TCM syndrome (Zhang et al., 2020). Mouse is the commonly used to the models mentioned above, however, it is increasingly accepted that the use of larger animal models, especially dogs and pigs, can provide deeper insights in cancer research (Croker et al., 2009).
The application of molecular tools and genetic strategies has aided the advancement of cancer research, and the cellular and animal models of CRC are being continually improved. Further understanding of the genetic and epigenetic events in CRC, including the alterations in molecular networks associated with the initial stages of development, are facilitated by high-resolution approaches.
Although CRC research has advanced immensely in recent years, several clinical issues remain to be resolved to date, which is partly attributed to the absence of suitable preclinical research models. The application of in vivo and in vitro models in CRC research, combined with advanced scientific techniques for simulating a more realistic tumor environment in vivo and in vitro, can help replicate the complex scenarios of tumor occurrence and development, identify novel therapeutic approaches for inhibiting tumor growth, and elucidate the molecular mechanisms underlying tumor formation.